I. Field of the Invention
This invention relates to the field of cancer therapy. The invention also relates to screening methods for identifying pharmacologically active compounds that may be useful for treating proliferative diseases. More particularly, the inventors have identified non-nucleoside molecule compounds that interact with specific DNA structures and which inhibit human telomerase.
II. Description of Related Art
Cancer, which is a cell proliferative disorder, is one of the leading causes of disease, being responsible for 526,000 deaths in the United States each year (Boring et al., 1993). For example, breast cancer is the most common form of malignant disease among women in Western countries and, in the United States, is the most common cause of death among women between 40 and 55 years of age (Forrest, 1990). The incidence of breast cancer is increasing, especially in older women, but the cause of this increase is unknown. Malignant melanoma is another form of cancer whose incidence is increasing at a frightening rate, at least sixfold in the United States since 1945, and is the single most deadly of all skin diseases (Fitzpatrick, 1986).
One of the devastating aspects of cancer is the propensity of cells from malignant neoplasms which disseminate from their primary site to distant organs and develop into metastatic cancers. Animal tests indicate that about 0.01% of circulating cancer cells from solid tumors establish successful metastatic colonies (Fidler, 1993). Despite advances in surgical treatment of primary neoplasms and aggressive therapies, most cancer patients die as a result of metastatic disease. Hence, there is a need for new and more efficacious cures for cancer.
The ends of chromosomes have specialized sequences, termed telomeres, comprising tandem repeats of simple DNA sequences. Human telomeres consist of the sequence 5xe2x80x2-TTAGGG (SEQ ID No. 1) (Blackburn, 1991; Blackburn et al., 1995). Telomeres have several functions apart from protecting the ends of chromosomes, the most important of which appear to be associated with senescence, replication, and the cell cycle clock (Counter et al., 1992). Progressive rounds of cell division result in a shortening of the telomeres by some 50-200 nucleotides per round. Almost all tumor cells have shortened telomeres, which are maintained at a constant length (Allshire et al., 1988; Harley et al., 1990; Harley et al., 1994) and are associated with chromosome instability and cell immortalization.
The enzyme telomerase adds the telomeric repeat sequences onto telomere ends, ensuring the net maintenance of telomere length in tumor cells commensurate with successive rounds of cell division. Telomerase is a DNA polymerase with an endogenous RNA template (Feng et al., 1995), on which the nascent telomeric repeats are synthesized. A significant recent finding has been that approximately 85-90% of all human cancers are positive for telomerase, both in cultured tumor cells and primary tumor tissue, whereas most somatic cells appear to lack detectable levels of telomerase (Kim et al., 1994; Hiyama et al., 1995a). This finding has been extended to a wide range of human tumors (see, for example, references Broccoli, 1994 and Hiyama et al., 1995b) and is likely to be of use in diagnosis.
Human telomerase has been proposed as a novel and potentially highly selective target for antitumor drug design (Feng et al., 1995; Rhyu et al., 1995; Parkinson, 1996). Studies with antisense constructs against telomerase RNA in HeLa cells show that telomere shortening is produced, together with the death of these otherwise immortal cells (Feng et al., 1995). Sequence-specific peptide-nucleic acids directed against telomerase RNA have also been found to exert an inhibitory effect on the enzyme Norton et al, 1996).
Among chemical agents, 2,6-diamido-anthraquinones have been reported as DNA-interactive agents (Collier and Neidle, 1988; 1992; Agbandje et al., 1992). These compounds have been shown to act as selective DNA triplex interactive compounds (Fox et al., 1995; Haq et al., 1996), with reduced affinity for duplex DNA and only moderate conventional cytotoxicity in a range of tumor cell lines. A carbocyanine dye, 3,3xe2x80x2-diethyloxadicarbocyanine (DODC,), has been reported to bind dimeric hairpin G-quadruplex structures (Chen et al., 1996).
This invention describes a novel class of non-nucleoside molecules that are telomerase inhibitors. These compounds have demonstrated their ability to interact with telomeres which form structures called the G-quadruplex structures. As telomeres are involved in controlling the cell cycle, cell replication and aging, these inhibitors of telomerase prevent uncontrolled cell growth and the immortality of tumor cells.
The present invention has demonstrated for the first time that a non-nucleoside, small molecule can target the G-quadruplex structure and can act as a telomerase inhibitor. Accordingly, methods have been developed that identify these classes of compounds and several inhibitors identified.
Compounds such as those described here, which interact selectively with G-quadruplex structures and inhibit telomerase, are expected to be useful as inhibitors of the proliferation of cells that require telomerase to maintain telomere length for continued growth. The invention thus relates to novel methods for identifying compounds that will be useful in this regard, and also includes new classes of telomerase inhibitors. In this regard, several perylene compounds and carbocyanines have been shown to interact with G-quadruplex structures. Since telomerase appears to be found almost exclusively in tumor cells, these agents are contemplated to be useful as antitumor agents.
In one aspect of the invention, compounds that act as telomerase inhibitors have been identified. It has been found that compounds that bind to the human G-quadruplex structure inhibit the human telomerase. The identification of such G-quadruplex interactive agents is an efficient approach for identifying human telomerase inhibitors. Methods for identifying these G-quadruplex interactive agents include identifying compounds whose three-dimensional structure is complementary to that of the G-quadruplex structure. Another method for identifying G-quadruplex interactive compounds is to identify compounds that interact with G-quadruplexes using such methods as dye displacement or melting points of G-quadruplex/compound hybrids.
More particularly, candidate compounds that inhibit telomerase activity are identified by first obtaining the three-dimensional structure of a compound that might interact with the G-quadruplex.selected compound. The complementarity of the compound to human telomere DNA G-quadruplex is then determined. If there is a high degree of complementarity, telomerase inhibition activity is indicated.
Alternatively, one can contact a telomerase inhibitor candidate compound with human DNA G-quadruplex; and then determine the melting point of the human DNA G-quadruplex. The inventors have found that an increase in melting point of the quadruplex indicates telomerase inhibitory activity of the compound.
Additionally, telomerase inhibitors may be identified by first preparing a DNA G-quadruplex/dye complex with a dye intercalated into the G-quadruplex; then contacting complex with a telomerase inhibitor candidate. Displacement of the dye in the complex identifies the candidate as a telomerase inhibitor.
Yet another aspect of this invention is to provide non-nucleoside inhibitors of telomerase. Using the disclosed screening methods, compounds have been identified that bind to human G-quadruplex structures. The invention includes perylene compounds, exemplified by N,Nxe2x80x2-bis(2-dimethylaminoethyl)-3,4,9,10-perylenetetracarboxylic acid diimide that are useful telomerase inhibitors. Novel compounds such as N,Nxe2x80x2-bis(2-piperdinoethyl)-3,4,9,10-perylenetetracarboxylic acid diimide are also within the scope of the invention.
A preferred G-quadruplex structure is formed from the sequence d(AGGGTTAGGGTTAGGGTTAGGG) (SEQ ID No. 2) or the sequences d(TTAGGG)4 (SEQ ID No. 1), d(TAAGGGT)4 (SEQ ID No. 3), or d(TTAGGGTT)4 (SEQ ID No. 4) either alone or in the presence of a G-quadruplex interactive perylene diimide of general structure I. The structures were determined by NMR spectroscopy. Alternatively, one may determine the three-dimensional structure of potential G-quadruplex interactive agents by x-ray diffraction or molecular mechanics calculations. Preferred programs for determining the degree of complementarity between the potential G-quadruplex interactive agent and these G-quadruplex structures include DOCK, autoDOCK, AMBER and SYBYL. The preferred methods for generating orientations between the potential G-quadruplex interactive agents and these G-quadruplex structures are manual and using the DOCK or autoDOCK programs. The cutoff for determining the likelihood that the orientation of the potential G-quadruplex interactive agent and the G-quadruplex structure have sufficient chemical interaction to form a complex is roughly 75% of the favorable intermolecular interaction energy, calculated with the above programs, of the perylene diimide 2-d(TTAGGG)4 (SEQ ID No. 1) complex structure as determined by NMR spectroscopy.
Preferred G-quadruplex structures are those formed by the sequences d(TTAGGG)4 (SEQ ID No. 1), d(AATGGGT)4 (SEQ ID No. 5) and d(TTAGGGTT)4 (SEQ ID No. 4). Several methods of determining the interaction of potential G-quadruplex interactive agents with these structures include UV/VIS spectroscopy, in which the changes in the UV/VIS spectrum of the potential agent under more than a 10% change at the wavelength due solely to the ligand and which undergoes the most change, upon addition of an excess of the G-quadruplex structure; UV spectroscopy, in which the melting temperature of the G-quadruplex structure as determined by a hyperchromicity transition at a given temperature range is increased by  greater than 50xc2x0 C. upon addition of an excess of the agent; UV/VIS spectroscopy in which addition of a potential G-quadruplex interactive agent to a complex of a G-quadruplex-interactive perylene diimide and a G-quadruplex produces a  greater than 25% change in the absorption of due to the G-quadruplex-interactive perylene diimine-G-quadruplex complex; UV/VIS spectroscopy in which addition of a potential G-quadruplex interactive agent to a complex of a G-quadruplex-interactive carbocyanine and a G-quadruplex produces a  greater than 25% change in the absorption of due to the G-quadruplex-interactive carbocyanine-G-quadruplex complex; NMR spectroscopy in which the melting temperature of the G-quadruplex as determined by the disappearance of the imino proton signals of the G-quadruplex is increase by  greater than 5xc2x0 C. in the presence of one- to two-equivalents of the agent; NMR spectroscopy in which the interaction of the agent with the G-quadruplex structure is determined by the shift of at least one of the imino protons of the G-quadruplex by  greater than 0.01 ppm upon addition of one- to two-equivalents of the agent; fluorescence spectroscopy in which the fluorescence emission spectrum of the agent undergoes a shift of  greater than 5 nm and/or a change in intensity of  greater than 25% upon the addition of an excess of the G-quadruplex structure; fluorescence spectroscopy in which the fluorescence emission spectrum of a G-quadruplex-interactive perylene diimide-G-quadruplex complex undergoes a  greater than 25% change upon the addition of an excess of the agent; or fluorescence spectroscopy in which the fluorescence emission spectrum of a G-quadruplex-interactive carbocyanine-G-quadruplex complex undergoes a  greater than 25% change upon the addition of an excess of the agent.
The preferred embodiments of the invention as it related to one class of G-quadruplex interactive telomerase inhibitors are compounds of the structure I in which 
in which R1 and R4 are independently taken from the set of sub-structures given by the formula -L-A in which L is a linking group taken from the set consisting of: 
where n is 1, 2, or 3; and each R5 is independently taken from the set H, Me, OH, or OMe; 
where R5 is as before and Y is taken from the set O, S, SO, SO2, NH, NMe, NCOMe; 
where R5 and Y are as before and X is taken from the set CH2, O, S, SO, SO2, NH, NMe, NCOMe; 
where R6, R7, R8, and R9 are independently taken from the set consisting of H, OMe, OEt, halo, or Me;
or a bond;
and A is taken from the set consisting of: 
where m is 0-5 and each R6 is taken from the set consisting of halo, NH2, NO2, CN, OMe, SO2NH2, amidino, guanidino, or Me; 
where o is 0 or 1; p is 0, 1, or 2; q is 1 or 2 such that o+q is either 2, in which case a pyrrolidine or pyrrole ring is indicated, or 3, in which a piperidine or pyridine ring is indicated; r is 0, 1, 2, or 3; R7 is H or Me; each R8 is independently taken from the set consisting of Me, NO2, OH, CH2OH, halo, or when r is 2 or 3, two adjacent R8 substituents may be together taken as xe2x80x94(CHxe2x95x90CH)2xe2x80x94 or xe2x80x94(CH2)4xe2x80x94 such as to form an annulated six-membered ring; 
where each R9 is independently taken from the set consisting of H, Me, or both R9 can taken together be xe2x95x90O; s is equal to 0 or 1; and Z is taken from the set consisting of CH2, O, NH, NMe, NEt, N(Me)2, N(Et)2, or NCO2Et; 
where Q is either N, CH, NMe, or NEt; X is either O, S, NH, NMe or NEt; R10 and R11 are independently taken from the set consisting of H, Me, CH2CO2Et, or R10 and R11 taken together consist of xe2x80x94(CHxe2x95x90CH)2xe2x80x94 or xe2x80x94(CH2)4xe2x80x94; 
where t is equal to 1, 2, 3, or 4; u is equal to 0, 1, 2, 3, or 4, and each R12 is individually taken from the set consisting of Me, or OH; 
OH, CO2R13, CON(R13)2, SO3H, SO2N(R13)2, CN, CH(CO2R13)2, CH(CON(R13)2)2, N(R13)2, or N(R13)3 where R13 is either H, Me, Et, or CH2CH2OH;
R2, R2xe2x80x2, R2xe2x80x3, R2xe2x80x3; R3, R3xe2x80x2, R3xe2x80x3, R3xe2x80x2xe2x80x3 are each independently taken from the set H, OMe, halo, or NO2.
In addition, this invention includes the development of other G-quadruplex interactive telomerase inhibitors compounds derived from structure I, having the following structures: 
The preferred embodiment of the invention as it relates to another class of G-quadruplex interactive telomerase inhibitors are compounds of the general structure II: 
in which C is either a bond, xe2x80x94CHxe2x95x90CHxe2x80x94, xe2x80x94(CHxe2x95x90CH)2xe2x80x94, xe2x80x94(CHxe2x95x90CH)3xe2x80x94, p-phenylene, o-phenylene, p-phenylene-CHxe2x95x90CHxe2x80x94, or o-phenylene-CHxe2x95x90CHxe2x80x94; B is O, S, or NR, and R is either Me or Et.
In addition, this invention includes the development of another G-quadruplex interactive telomerase inhibitor compound derived from structure H, having the following structure: 